Project Summary Eukaryotic cells must be able to maintain balance between the amount of protein entering the secretory pathway and the capacity to process, fold and traffic these proteins through the endoplasmic reticulum. Excessive protein synthesis can cause proteins to unfold and aggregate in the ER causing ER stress. To survive the cells launch a signal transduction cascade called the Unfolded Protein Response (UPR). The UPR leads to increases in protein folding capacity by upregulating chaperones, also prevents new proteins from entering the ER by lowering the rate of global translation, and the UPR also helps clear unfolded proteins from the ER by increase proteolysis and autophagy. Numerous human disease are linked to ER-stress including, Alzheimer?s and other neurodegenerative diseases, type II diabetes, fatty liver disease, spinal cord injury, among others. Multiple drugs have been developed that modulate UPR signaling; among the most promising are inhibitors of eIF2 alpha phosphatases (including salubrinal and sephin1). These drugs have shown great promise in delaying onset and prolonging survival in mouse models of Amyotrophic lateral sclerosis (ALS), while sephin1 has been recently approved as an orphan drug for treating Charcot-Marie-Tooth disease 1B (CMT). The eIF2 alpha phosphatase complexes are comprised of a catalytic subunit PP1 and either of two regulatory subunits, GADD34 and CReP, and are potent inhibitors of one branch of the UPR. This project uses the zebrafish model system to investigate the molecular mechanisms of ER-stress induced apoptosis. In aim 1 we will take a genetic approach and seek to understand how levels of GADD34 and CReP are regulated by ER stress. Our preliminary data indicates that GADD34 mRNA rapidly increases and remains elevated after ER-stress, while CReP transiently increases following ER stress and then declines to levels below baseline after prolonged stress. We will identify which of the branches of UPR signaling are required for this dynamic expression pattern. In addition, we will utilize CRIPSR-CAS9 genome editing for reverse-genetic analysis of the function of GADD34 and CReP. In aim 2 we will then focus on two major stress-induced phenotypes in zebrafish, ER-stress induced apoptosis in the developing caudal fin and ER-stress induced accumulation of fatty acids in the liver (steatosis). This will provide us with a highly tractable model of ER-stress induced cell death and ER-stress induced metabolic disorders. Our preliminary data indicates that inhibition of GADD34 and CReP protects fin epidermal cells from ER-stress induced apoptosis, but worsens fatty liver symptoms in the liver. In aim 3 we will examine the assembly of the active eIF2 alpha phosphatase by determining the amino acids required for in vivo function. In our preliminary data experiments we mapped the minimal PP1-binding domain of CReP and GADD34 and generated a suite of point mutants to map residues critical for function. Using zebrafish will allow us to combine reverse genetics with in vivo cell biology, and will provide an ideal model to test the function of GADD34 and CReP and shed light onto the mechanism of action of clinically relevant drugs that target these proteins.